JP7529577B2 - Lactobacilli for Skin Care - Google Patents

Description

DSMZ DSM15316 DSMZ DSM15313

The present invention relates to microorganisms for use in the treatment and/or prevention of skin conditions by topical application, the microorganisms being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316). In particular, the microorganisms are intended for use in the treatment and/or prevention of compromised skin barrier function, inflammatory skin conditions and/or the proliferation of pathogenic microorganisms. The present invention further relates to particulate preparations comprising the microorganisms and a carrier as well as pharmaceutical or cosmetic compositions or products comprising these microorganisms. Cosmetic uses of these microorganisms or compositions for application to the skin as well as cosmetic methods, in particular for improving the appearance of the skin and/or preventing body odor, are also provided.

Probiotics are well known to improve gut health, attributed to enhanced gut-brain axis interactions, reduce lactose intolerance, are beneficial in treating vaginal or urogenital infections or prevent inflammatory bowel disease. Lactobacillus sp. in particular have a safe and long tradition in food fermentation processes. In contrast to the gut, little is known about the interactions between the epidermis, skin microbiota and environmental microbes.

Today, more and more research is showing that probiotics have other health-promoting effects, including skin health. Bifidobacteria and lactobacilli are the most common types of probiotics used, and their use in skin care has been actively developed over the past few years.

However, not all probiotic strains show beneficial effects on the skin, and single strains are rarely mentioned. Moreover, if at all, a balance needs to be achieved between the anti-inflammatory or other effect as an active ingredient in a cosmetic composition and not causing any pro-inflammatory side effects by the microorganism itself.

The skin is constantly exposed to bacteria, microorganisms and potential pathogens. To avoid infection or invasion by these pathogenic bacteria, the skin has developed a multifaceted defense strategy. Besides the physical and chemical barrier of the stratum corneum, it is equipped with a constitutive and inducible AMP barrier that is essential in this context (Harder et al., 2013; Wiesner and Vilcinskas, 2010).

Antimicrobial peptides (AMPs) are a diverse and abundant group of molecules. They are characterized by their small size, amino acid composition, amphipathicity and/or cationic charge. Based on their amino acid composition they are often divided into characteristic subgroups. The diversity of their antimicrobial potential and mechanism of action is as diverse as their structure and composition. They can be detected in various tissues and cell types of invertebrates, animal species and plants (Brogden 2005; Zasloff 2002). They serve as a first line of defense in human skin, since they are active against a wide range of gram-positive and gram-negative bacteria, fungi, eukaryotic parasites and/or viruses (Brown and Hancock 2006).

AMP expression can be upregulated in response to various environmental stimuli such as contact with pathogens or intracellular inflammatory pathways. Commensal and pathogenic bacteria differ in their ability to induce AMP expression, activating different signaling pathways in human skin to innate human immunity. Little is known about the specific interactions between the two. For example, β-defensins are upregulated by various stimuli of inflammation and/or infection such as interleukin-1 or bacterial lipopolysaccharide (Chung and Dale, 2004). US Patent No. 5,999,633 discloses that LPS from Vitreoscilla filiformes, which contains lipid A, has the ability to stimulate the expression of AMPs in keratinocytes without having pathogenic activity.

It is known that the skin microbiome may play an important role in various skin diseases and/or health conditions. The complexity and inter/intra-individual variation of the microbiome as well as difficult problems in characterizing, classifying and analyzing it pose challenges in understanding the interactions and developing topical therapeutic and/or diagnostic applications and/or treatments that help to influence, interact and/or support the microbiome. Nevertheless, the microbiome and microbiome-related diseases are becoming an increasingly important aspect, inducing the need to develop microbiome-related treatments of diseases and health conditions.

For example, atopic dermatitis (AD) is a multi-functional complex disorder with various genetic risks but prominent environmental triggers. AD is associated with physical barrier defects and/or dysbiosis (microbial imbalance). Patients show suppression of various AMPs such as cathelicidin or β-defensins or alterations in T-cell homeostasis. The combination of antimicrobial and physical barrier defects drives dysbiosis and produces further disturbances in the cellular immune balance that is again necessary to establish equilibrium with the external environment (Ong et al., 2002; Palmer et al., 2006; Sakaguchi et al., 2010).

Furthermore, other common human skin diseases such as rosacea or psoriasis are also associated with dysregulation of AMP-expression. Specific manipulation and induction of these endogenous peptides can restore, maintain and/or improve a balanced skin microbiome. This represents a new strategy to create skin benefits in a wide range of applications.

Furthermore, it is known that Staphylococcus aureus is frequently found in eczematous skin lesions of AD patients and is important and likely causative of the exacerbation of the disease symptoms. Skin that has been irritated or damaged by scratching, sunburn or other trauma is more likely to become infected. For example, dry skin is also easily damaged. Avoidance of this type of infection represents the second most relevant starting point for developing alternative therapeutic treatments.

From the state of the art several publications are known dealing with the various beneficial effects of probiotics for skin care and skin health. Lactobacillus preparations have been used for various purposes in cosmetic applications, mainly based on health benefits for the skin, body, hair or nails, but also relating to technical applications. Most of the compositions described contain mixtures of different live or non-viable probiotic strains, preferably used in combination with fermented plants, food or chemical agents. On the other hand, the beneficial effects of single pure cultured strains have rarely been shown.

In US Pat. No. 5,399,434, the use of Lactobacillus reuteri, Lactobacillus rhamnosus and Bifidobacterium longum for cosmetic applications and topical skin treatments is described.

Patent document 3 discloses an aqueous lotion composition for topical application containing an extract derived from the fermentation of probiotic microorganisms, including Lactobacillus or Bifidobacterium in general.

The beneficial effect of microorganisms for skin barrier function was described in US Pat. No. 5,399,633. The cosmetic use of at least one probiotic microorganism/fraction in combination with hesperidin to prevent the deterioration of the skin barrier function and/or to strengthen the skin barrier function is disclosed.

The possibility of inducing β-defensin in skin cells by two strains of L. plantarum is described in US Pat. No. 5,999,433. Antibacterial effects on the skin and improvements in acne and sensitive skin have been demonstrated.

WO 02/06331 describes the cosmetic use of lactic acid bacteria and/or compositions containing them for the topical treatment and/or prevention of skin infections, chronic wounds or skin diseases caused by pathogenic microorganisms such as S. aureus or Pseudomonas aeruginosa. Efficacy is caused by specific bacterial coaggregation of the lactic acid bacteria with the pathogens.

Patent document 7 discloses a pharmaceutical preparation comprising a combination of various viable α-Streptococcus strains and Lactobacillus strains that is useful for the treatment of Staphylococcus-induced infections of human skin.

WO 02/06339 discloses a formulation comprising at least two strains of lactic acid bacteria selected from a pool including L. plantarum for the treatment and/or prevention of stress-induced inflammatory disorders. The claimed effect for topical application is limited to severely damaged skin, i.e. ulcers or burn wounds.

Topical application of Lactobacillus plantarum to severe human burns has been described to improve tissue repair and prevent infection (Peral et al., 2009).

WO 02/06331 discloses a wide range of lactic acid bacteria, including Lactobacillus plantarum, that produce active substances, in particular bacteriocins or bacteriocin-like substances. It is suggested that these strains be used in the prevention and treatment of skin infections or inflammatory skin diseases caused by bacteria, such as atopic dermatitis or acne.

WO 02/06336 describes a composition comprising Lactobacillus plantarum CNCM1-4026 for use in preventing or treating skin infections. In particular, it was found that concentrated supernatants recovered after Lactobacillus fermentation inhibit or retard the growth of various Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus or Staphylococcus epidermidis. Only soluble bacterial components obtained from the growth medium were used, not whole bacterial cells.

The inhibitory effect of Staphylococcus aureus and various lactic acid bacteria, including Lactobacillus plantarum, has been observed using cell-free supernatants and extracts (Yong et al., 2015).

WO 02/06331 relates to microorganisms for use in the treatment and prevention of inflammation in the oral cavity, in particular for treating or preventing gingivitis and periodontitis. Among the microorganisms disclosed, Lactobacillus plantarum HEAL19 has been shown to have an anti-inflammatory effect on monocytes. However, it cannot be concluded from WO 02/06331 that such an effect extends to epidermal skin cells outside the oral cavity, i.e. non-mucosal keratinocytes. The same effect observed in monocytes cannot be expected to apply to skin, since non-mucosal skin has a completely different structure, especially with regard to the protective layer (e.g. stratum corneum) and the associated strong barrier function. Moreover, the epidermis is a non-vascularized tissue that does not contain any monocytes or related cell types at all.

US Patent Application Publication No. 2009/0035294(A1) International Publication No. 2013153358 International Publication No. 2015048511 International Publication No. 2009031106 International Publication No. 2005091933 US Patent Publication No. 2014186409 International Publication No. 2010056198 International Publication No. 2005077391 Korean Patent Publication No. 2011134151 International Publication No. 2016023688 International Publication No. 2017125447(A1)

It was an object of the present invention to provide a probiotic bacterial strain that helps to treat and/or prevent skin conditions, in particular impaired skin barrier function, inflammatory skin conditions and the growth of pathogenic microorganisms.

It was a further object of the present invention to provide probiotic bacterial strains that are useful in cosmetic applications, in particular those that can improve the appearance of the skin and prevent body odor.

In particular, it was also an object of the present invention that the strain provided by the present invention has one or preferably two, several or all of the effects selected from strengthening the skin barrier, soothing the skin and reducing or preventing inflammation, enhancing the immune defense of the skin by upregulating AMP, delaying skin aging, inhibiting the growth of pathogenic microorganisms on the skin and preventing infectious diseases.

Furthermore, the provided strains should not have undesirable side effects, such as pro-inflammatory effects, when used on the skin.

The above objectives are met by a microorganism or a mixture comprising or consisting of two microorganisms for use in the treatment and/or prevention of skin conditions by topical application, the microorganisms being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316).

According to the invention, in all embodiments, a mixture of either or both of the Lactobacillus plantarum strains HEAL19 and HEAL99 can be used. Thus, the expression "a microorganism or a mixture comprising or consisting of two microorganisms" refers to either one of the two strains present or both strains present.

It was known from the prior art that some probiotic bacteria can improve certain aspects of skin conditions and skin appearance, but not all bacterial strains provide beneficial effects on the skin and some may even be harmful. Furthermore, beneficial effects have been shown in the case of application of microorganisms to severely injured or burned skin as well as to mucous membranes in the oral cavity. However, these effects cannot be transferred or compared to application to non-mucosal skin with strong barrier functions. The presence of a skin barrier is a prerequisite for inducing the effects described in the present invention.

It has now been found that Lactobacillus plantarum HEAL 19 and HEAL 99 offer a unique combination and a comprehensive spectrum of activity that not only decisively improves the clinical symptoms and cosmetic appearance of the skin, but also supports healthy skin and helps to prevent disorders. The topical application of these strains is shown to offer, in addition to their anti-inflammatory potential, a combination of skin barrier support through the induction of various AMPs, as well as strengthening the physical skin barrier through multiple pathways. Furthermore, a growth inhibitory effect of Staphylococcus aureus could also be shown for the described Lactobacillus plantarum strains.

In particular, it has been shown that heat-treated Lactobacillus containing both water-soluble and water-insoluble components can improve skin health. The bacterial suspension has the potential to strengthen the skin's physical barrier by reducing trans-epidermal water loss (TEWL) and/or increasing components related to natural moisturizing factors. At the same time, the production of various AMPs in the skin is induced, enhancing the skin's self-defense. Potential skin infections can be prevented by inhibiting the growth of pathogens such as S. aureus. Furthermore, L. plantarum HEAL19 and HEAL99 have shown anti-inflammatory effects.

In conclusion, L. plantarum HEAL 19 and 99 strains show high and comprehensive potential as agents to modify skin properties and skin health when applied topically.

In the context of the present invention, the term "skin" refers to skin that is non-mucosal skin. In particular, mucous membranes found in body cavities such as the oral cavity, stomach, intestines, bronchi, anus or vagina are not encompassed by the term "skin". Thus, the term "topical application" refers to the application of agents and/or formulations to the skin and excludes any application inside a body cavity encompassed by a mucous membrane. Preferably, the skin still retains its protective function that has not been significantly weakened, for example by severe injury or burns.

"By topical application" in the context of the present invention means that the microorganism described herein, or a preparation, pharmaceutical or cosmetic composition or product containing said microorganism, is administered directly by topical application to the skin, i.e. directly to the surface of the skin.

"Skin condition" in the context of the present invention refers to any condition of the skin, preferably associated with a skin pathology, other than wounds or burns. "Skin condition" preferably relates to a condition characterized by discomfort, such as itching, or represents a cosmetic problem, such as peeling, dryness, redness, rash, acne, oiliness or body odor, caused by bacterial growth. Examples of skin conditions are given below:

The strain Lactobacillus plantarum HEAL19 was transferred on 27 November 2002 to the Leibniz Institut Deutche Sammlung für Mikroorganismen und Zellkulturen GmbH (DSMZ), Infoffenstr. 7B, 38124, Sweden, under the terms of the Budapest Treaty. It has been deposited in Braunschweig, Germany under the accession number DSM 15313. The strain Lactobacillus plantarum HEAL99 has been deposited under the Budapest Treaty by Probi AB, Soelvegatan, 41, 223 70 Lund, Sweden on November 27, 2002 at the Leibniz-Institut Deutsche Sammlung für Microorganismen und Zellkulturen, Imhoffenstraße 7B, 38124 Braunschweig, Germany under the accession number DSM 15316.

In a preferred embodiment, the microorganism has been subjected to a heat treatment, which preferably results in the inactivation, attenuation or death of the microorganism. Preferably, the heat-treated microorganism has an intact physical structure.

The heat treatment may be carried out by ultrasound, UV irradiation or heating. Preferably, it is carried out by heating to a temperature of 60-121°C for 1 second to 120 minutes, for example 20 seconds to 120 minutes. In a preferred embodiment, the heat treatment is preferably carried out at between 70-100°C for 20 seconds to 15 minutes using an industrial pasteurizer. Preferably, the microorganisms are killed and therefore can no longer reproduce. Preferably, the heat treated microorganisms still have an intact physical structure. This means that the cell structure has not been destroyed by the heat treatment and the microorganisms can be delivered containing metabolic products. Advantageously, upon heat treatment, the microorganisms typically maintain their ability to provide beneficial effects for treating skin conditions as described herein and due to the fact that viable microorganisms are not necessary for the purposes of the present invention, it is possible to combine them with preservatives. This is very important for most applications, since pharmaceutical and cosmetic products contain preservatives to prevent bacterial or fungal growth in the product upon repeated opening and closing of the container, which is often the case while the product is being used up.

The skin condition to be treated and/or prevented is preferably a compromised skin barrier function, an inflammatory skin condition and/or the proliferation of pathogenic microorganisms.

"Skin barrier" in the context of the present invention refers on the one hand to the physical and chemical barrier of the stratum corneum, which prevents substances from entering the body and protects against drying out, but on the other hand also to the induced AMP barrier, which together with other chemical properties such as pH, provides protection against colonization or invasion by microbial pathogens. Thus, "reduced skin barrier function", which also includes "reduced skin barrier function", means that the normal or healthy skin structure and/or function is disturbed to such an extent that it can no longer provide the above-mentioned protection or defense to a satisfactory degree. However, preferably, such a disturbance is not an injury, such as a cut or severe burn.

An "inflammatory skin condition" is a condition caused by a biological response of the skin to a harmful stimulus (pathogenic toxin), such as an irritant or pathogenic microorganism, and typically results in redness, swelling, heat and/or pain in the affected area. To assess the inflammatory state, inflammatory mediators such as inflammatory cytokines or chemokines, e.g., interleukin 8 (IL-8), can be measured in the affected tissue.

"Pathogenic microbial growth" such as bacteria or fungi on or in the skin can result in infection and/or inflammation, but also cosmetic problems such as rashes, acne or body odor. In particular, "pathogenic microbial growth" refers to an increase in the amount of pathogenic microorganisms on or in the skin above the levels associated with a healthy microbiome. In particular, it refers to a microbial infection.

The above three aspects are closely linked. For example, a decrease in barrier function can result in the invasion of pathogenic microorganisms or harmful substances, which in turn cause inflammation.

In a preferred embodiment, the microorganism is
(a) an agent for strengthening the skin barrier function, and/or (b) an agent for reducing transepidermal water loss, and/or (c) an agent for inducing the expression of filaggrin, and/or (d) an agent for increasing components related to natural moisturizing factors, and/or (e) an agent for providing anti-inflammatory activity, in particular an agent for reducing and/or suppressing inflammatory parameters, and/or (f) an agent for providing anti-inflammatory activity induced by chemokines, and/or (g) an agent for providing anti-inflammatory activity induced by external pathogenic toxins, such as pathogenic microorganisms, in particular Staphylococcus aureus, and/or air pollutants, and/or ultraviolet radiation and/or surfactants, and/or (h) an agent for inhibiting the growth and/or invasion and/or infection of pathogenic microorganisms, in particular Staphylococcus aureus, and/or (i) an agent for inducing the expression of antimicrobial peptides (AMPs), and/or (j) an agent for maintaining and/or establishing and/or repairing a healthy state of the skin microbiome, and/or (k) an agent for improving the immune response of the skin.

It has been found that the Lactobacillus plantarum strains HEAL19 and HEAL99 are able to strengthen the skin barrier in various ways. For example, they are able to reduce transepidermal water loss (TEWL) by inducing the expression of filaggrin, which is essential for the regulation of epidermal homeostasis, especially moisturization. Furthermore, the strains have been shown to increase components related to natural moisturizing factors, which likewise play an important role in proper stratum corneum hydration and barrier homeostasis. On the other hand, in a further aspect that contributes to the strengthening of the skin barrier function, the strains are able to induce the expression of antimicrobial peptides (AMPs), which result in increased protection against the invasion of pathogenic microorganisms, such as bacteria and fungi.

"Induction" or upregulation of expression of a gene in the context of the present invention may be determined using a reference gene, e.g. a so-called housekeeping gene, whose expression does not change significantly in different tissues or under different conditions. Genes suitable as reference genes in human cells are known to the skilled artisan. In the experimental procedure below, the determination of induction or upregulation of expression using a reference gene for normalization is described.

It has further been found that Lactobacillus plantarum strains HEAL19 and HEAL99 can reduce inflammatory mediators such as IL-8 in the skin, which are released in response to various stimuli, such as external toxins, e.g., pathogenic microorganisms or pollutants. Thus, they can reduce inflammatory responses and associated symptoms.

Furthermore, Lactobacillus plantarum strains HEAL19 and HEAL99 were shown to have an inhibitory effect on the growth of pathogenic microorganisms, especially Staphylococcus aureus. They induce the expression of antimicrobial peptides (AMPs) and thus can enhance the immune response of the skin and maintain or establish a healthy skin microbiome. A healthy microbiome is represented by a balanced colonization of certain microorganisms that are not harmful or even beneficial. Harmful effects can occur if this balance shifts towards a state known as microbial dysbiosis.

Due to the diverse but interrelated effects described above, Lactobacillus plantarum strains HEAL19 and HEAL99 are useful for the prevention and/or treatment of multiple pathologies as well as improving other cosmetic aspects such as skin appearance and body odor.

In a preferred embodiment, the skin condition is selected from the group consisting of atopic dermatitis, microbial infection, dry skin, itchy skin, sensitive skin, atopic skin, skin inflammation, microbial dysbiosis, rosacea, psoriasis, rash and acne.

The present invention also relates to a preparation comprising or consisting of a microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316) and a carrier, the preparation being a particulate preparation, preferably a granule or a powder.

It has been found to be particularly advantageous for the purposes of the present invention to deliver the microorganisms in the form of a particulate preparation with a carrier. Particulate preparations, especially dry granules or powders, are more stable than, for example, suspensions and have a significantly longer shelf life when stored prior to application. Thus, no preservatives are required to stabilize the preparations, and no special storage measures, such as refrigeration, are necessary. Even over longer storage periods, the preparations do not show discolouration or other signs of deterioration. Furthermore, particulate preparations are homogeneous in terms of distribution of the microorganisms, without undesirable effects such as precipitation or agglomeration affecting the homogeneity. Thus, particulate preparations can also be easily and efficiently processed into pharmaceutical or cosmetic compositions or products.

A "particulate preparation" in the context of the present invention is a preparation consisting of well-defined particles. Preferably, the particles have a uniform size and an even distribution of microorganisms and carriers, are dry and flowable and do not tend to form lumps. In particular, a particulate preparation may be a granule or a powder. Such a particulate preparation or, respectively, a granule or powder may comprise or consist of particles having an average particle size in the range of 0.1 to 200 μM, preferably 1 to 100 μM.

The particulate preparation according to the invention can be obtained by freeze-drying, spray-drying or granulating the microorganism together with a carrier.

In a preferred embodiment of the above preparation, the microorganism has been subjected to heat treatment and is preferably an attenuated or killed microorganism. However, as explained above, preferably the heat-treated microorganism still has an intact physical structure.

Advantageously, the microorganisms maintain their beneficial properties for the topical treatment of skin conditions upon heat treatment. Therefore, the preparations can be used in pharmaceutical or cosmetic products that contain preservatives, since viable microorganisms are not required.

The carrier used in the preparation according to the invention is a material that is suitable to be provided in particulate form, for example by freeze-drying, spray-drying or granulation. Furthermore, it is important that the carrier is a pharma- ceutically and cosmetically acceptable material.

In the above preparations, the carrier may be selected from the group consisting of polysaccharides, preferably inulin, starch, gum arabic, whey protein, skimmed milk powder and maltodextrin and combinations thereof, preferably maltodextrin.

In the context of the present invention, it has been found that the ratio between microorganisms and carrier is crucial for the properties of the preparation. If too little carrier, e.g. maltodextrin, is used, the preparation typically becomes hygroscopic, which can adversely affect its stability and increase the tendency to form lumps.

In a preferred embodiment, the ratio of microorganism to carrier in the above preparations is in the range of 1:9 to 3:7, preferably 1.5:8.5 to 2.5:7.5, and/or the preparations comprise 10-30% by weight of microorganism and 70-90% by weight of carrier, preferably 15-25% by weight of microorganism and 75-85% by weight of carrier, in each case relative to the total weight of the preparation.

The present invention also relates to a preparation according to any of the above embodiments for use in the treatment and/or prevention of a skin condition by topical application. In particular, the skin condition to be treated and/or prevented is a compromised skin barrier function, an inflammatory skin condition and/or a proliferation of pathogenic microorganisms, preferably the skin condition is selected from the group consisting of atopic dermatitis, microbial infections, dry skin, itchy skin, sensitive skin, atopic skin, skin inflammation, microbial dysbiosis, rosacea, psoriasis, rash and acne.

The present invention relates to a method for producing a preparation, preferably according to any of the above embodiments, which comprises or consists of a microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316) and a carrier, comprising:
(i) providing a microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316);
(ii) optionally subjecting the microorganism of step (i) to a heat treatment, preferably at a temperature in the range of from 60 to 121° C. for a period of from 1 second to 120 minutes;
(iii) combining the microorganism from step (i) or (ii) with a carrier and further processing the combination, preferably by freeze-drying, spray-drying or granulation, to obtain a granule or powder;
The present invention also relates to a method comprising the steps of:

The above process represents a highly advantageous way of processing microorganisms for the purposes of the present invention since the resulting preparations are, as described above, highly stable and can be easily and efficiently processed into a wide range of pharmaceutical or cosmetic products.

According to a further aspect, the present invention relates to a pharmaceutical or cosmetic composition or a pharmaceutical or cosmetic product for topical application to the skin comprising one microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316) or comprising a preparation according to any of the above embodiments, wherein the total amount of microorganisms is sufficient to treat and/or prevent skin conditions, in particular inflammatory skin conditions, a compromised skin barrier function and/or the proliferation of pathogenic microorganisms, preferably the total amount of microorganisms is in the range of 0.01 to 5% by dry weight, preferably 0.02 to 1% by dry weight, for example 0.1 to 1% by dry weight, in each case relative to the total weight of the composition, and/or the total amount of microorganisms is at least 10 ⁸ per gram of the total composition, preferably 5×10 ⁸ cells or at least 10 ¹⁰ cells per gram, preferably 10 The present invention also relates to ^{pharmaceutical} or cosmetic compositions or pharmaceutical or cosmetic products for topical application to the skin, which contain a concentration of 10 to ^5x10 cells (depending on the type of composition, for example whether the composition is a semi-finished or finished product), for example in the range of ¹⁰ to 5x10 cells, preferably ¹⁰ to ¹⁰ cells per gram of total composition.

Surprisingly, it has been found that Lactobacillus plantarum HEAL 19 and Lactobacillus plantarum HEAL 99 are already able to provide the effects described herein at concentrations as low as 0.01 or 0.02% of the dry weight of the pharmaceutical or cosmetic composition or product.

For example, the Lactobacillus suspension or, more preferably, the particulate preparations described above can be used as an active ingredient for various pharmaceutical or cosmetic applications, such as topical leave-on or rinse-off formulations. Compositions containing an effective amount can be useful for treating skin disorders such as reduced skin sensitivity or dry skin, skin itching or atopic dermatitis. These compositions are suitable for continuous treatment of atopic skin as well as application to specific affected areas of the skin such as lesions, scratches or wounds. Furthermore, these compositions can be used routinely to maintain an intact barrier as well as a healthy skin microbiome.

In a preferred embodiment of the pharmaceutical or cosmetic composition or product, the microorganisms are subjected to a heat treatment, preferably attenuated or killed, but still with an intact physical structure. For the conditions of the heat treatment, the parameters defined above are appropriately applied.

Advantageously, microorganisms typically maintain their beneficial properties for the topical treatment of skin conditions upon heat treatment, and the pharmaceutical or cosmetic product may contain a preservative, since viable microorganisms are not required.

Thus, in a preferred embodiment, a pharmaceutical or cosmetic composition or product according to any of the embodiments described herein includes at least one preservative.

"Preservative" in the context of this invention refers to a substance that inhibits or inhibits microbial growth.

The present invention also relates to a pharmaceutical or cosmetic composition or product as described above for use in the treatment and/or prevention of skin conditions, in particular impaired skin barrier function, inflammatory skin conditions and/or the growth of pathogenic microorganisms, by topical application.

Since Lactobacillus plantarum strains HEAL19 and HEAL99 provide the above-mentioned benefits a)-k) when applied topically to the skin, compositions and products containing an effective amount of one or both strains are effective in treating and/or preventing multiple skin conditions and improving the appearance of the skin as described above.

The compositions and products according to the invention are intended for topical application to the skin and are therefore preferably in formulations suitable for topical application to the skin. Such formulations may be leave-on or rinse-off formulations.

In a further preferred embodiment, said pharmaceutical or cosmetic composition or product is selected from the group consisting of oil-in-water or water-in-oil emulsions, ointments, cremes, lotions and gels.

These compositions and products may contain further ingredients that provide suitable properties for application to the skin. Thus, pharmaceutical or cosmetic compositions or products are preferred that further comprise one or more ingredients selected from the group consisting of carriers, excipients and further active ingredients, preferably selected from maltodextrin, inulin, emollients and vegetable oils.

The present invention also relates to a method for producing a pharmaceutical or cosmetic composition or product, preferably a pharmaceutical or cosmetic composition or product according to any of the embodiments described herein, comprising:
(i) preparing a microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), preferably the microorganism is an attenuated or killed microorganism that has been subjected to a heat treatment and preferably has an intact physical structure, or a preparation according to any of the above embodiments or produced by the above method;
(ii) combining the microorganism or preparation of step (i) with one or more substances selected from carriers, excipients and further active ingredients to obtain a pharmaceutical or cosmetic composition or product;
The present invention also relates to a method comprising the steps of:

In a preferred embodiment of the method, in step (ii) at least one preservative is combined with the microorganism or preparation of step (i).

According to the present invention, the vegetable oil is selected from the group consisting of argan oil, chokeberry (seed) oil, avocado oil, peach (pit) oil, canola oil, nigella sativa oil, pumpkin (pumpkin seed) oil, wild rose (seed) oil, pomegranate seed oil, jojoba (liquid wax) oil, cacao/cocoa butter, wheat sprout oil, coconut/coconut butter, safflower oil, corn oil, camelina oil, flax seed oil, macadamia oil, raspberry seed oil, melon seed oil, thyme ... It may be selected from the group consisting of doughnut seed oil, passionflower seed oil, almond oil, neem oil, moringa oil, borage oil, olive oil, peanut oil, hazelnut oil, walnut oil, palm oil, papaya seed oil, parsley seed oil, seabuckthorn oil, castor oil, rice oil, sesame oil, shea butter/karite butter, sunflower oil, soybean oil, tamanu oil, evening primrose oil, grape seed oil, and cranberry seed oil.

According to the invention, Guerbet alcohols, which are primarily fatty alcohols having 6 to 18, preferably 8 to 10, carbon atoms, esters of linear C ₆ -C ₂₂ -fatty acids with linear or branched C ₆ -C ₂₂ -fatty alcohols or esters of branched C ₆ -C ₁₃ -carboxylic acids with linear or branched C ₆ -C ₂₂ -fatty alcohols are preferred. - esters with fatty alcohols, for example myristyl myristitate, myristyl palmitate, myristyl stearate, myristyl isostearate, myristyl oleate, myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate, cetyl stearate, cetyl isostearate, cetyl oleate, cetyl behenate, cetyl erucate, stearyl myristate, stearyl palmitate, stearyl stearate, stearyl isostearate, stearyl oleate, stearyl behenate, stearyl erucate, isostearyl myristate, isostearyl palmitate, isostearyl stearate, isostearyl Further suitable oil bodies may be selected from the group consisting of oleyl isostearate, isostearyl oleate, isostearyl behenate, isostearyl oleate, oleyl myristate, oleyl palmitate, oleyl stearate, oleyl isostearate, oleyl oleate, oleyl behenate, oleyl erucate, behenyl myristate, behenyl palmitate, behenyl stearate, behenyl isostearate, behenyl oleate, behenyl behenate, behenyl erucate, erucyl myristate, erucyl palmitate, erucyl stearate, erucyl isostearate, erucyl oleate, erucyl behenate and erucyl erucate. Esters of linear C ₆ -C ₂₂ -fatty acids with branched alcohols, in particular 2-ethylhexanol, esters of C ₁₈ -C ₃₈ -alkylhydroxycarboxylic acids with linear or branched C ₆ -C ₂₂ -fatty alcohols, in particular dioctyl malate, esters of linear and/or branched fatty acids with polyhydric alcohols (such as, for example, propylene glycol, dimer diols or trimer triols) and/or Guerbet alcohols, triglycerides based on C ₆ -C ₁₀ -fatty acids, liquid mono-/di-/triglyceride mixtures based on C ₆ -C ₁₈ -fatty acids, esters of C ₆ -C ₂₂ -fatty alcohols and/or Guerbet alcohols with aromatic carboxylic acids, in particular benzoic acid, C ₂ -C ₁₂ - esters of dicarboxylic acids with linear or branched alcohols having 1 to 22 carbon atoms or polyols having 2 to 10 carbon atoms and 2 to 6 hydroxyl groups, vegetable oils, branched primary alcohols, substituted cyclohexanes, linear and branched C ₆ -C ₂₂ fatty alcohol carbonates, for example Guerbet carbonates based on fatty alcohols having 6 to 18, preferably 8 to 10, carbon atoms, such as dicaprylyl carbonate (Cetiol® CC), benzoic acid with linear and/or branched C ₆ -C ₂₂ fatty acids. Also suitable are esters with alcohols (e.g. Finsolv® TN), linear or branched, symmetrical or asymmetrical dialkyl ethers having 6 to 22 carbon atoms per alkyl group, such as dicaprylyl ether (Cetyoll® OE), ring-opening products of epoxidized fatty acid esters with polyols, silicone oils (cyclomethicones, silicone methicone grades, etc.), aliphatic or naphthenic hydrocarbons, such as squalane, squalene or dialkylcyclohexanes and/or mineral oils.

In the compositions and products according to the invention, Lactobacillus plantarum strains HEAL19 and HEAL99 are preferably used in combination with one or more (further) substances for preventing, reducing or alleviating dry skin and/or itchy skin symptoms and/or one or more substances selected from the group consisting of one or more skin irritation reducing agents, in particular anti-inflammatory agents, physiological cooling agents and compounds which reduce redness, preferably said one or more additional substances being
(i) antipruritic compounds,
(ii) steroidal anti-inflammatory substances of the corticosteroid type, in particular hydrocortisone, hydrocortisone derivatives such as hydrocortisone 17-butyrate, dexamethasone, dexamethasone phosphate, methylprednisolone or cortisone,
(iii) non-steroidal anti-inflammatory substances, in particular oxicams such as piroxicam or tenoxicam, salicylates such as aspirin, disalcid, solprin or fendosal, acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin or clindanac, fenamates such as mefenam, meclofenam, flufenam or niflum, propionic acid derivatives such as ibuprofen, naproxen or benoxaprofen, pyrazoles such as phenylbutazone, oxyphenylbutazone, febrazone or azapropazone,
(iv) natural or naturally occurring anti-inflammatory substances which reduce redness and/or itching, in particular extracts or fractions from chamomile, aloe vera, Commiphora, Rubia, willow, willow herb, oat, calendula, arnica, St. John's wort, honeysuckle, rosemary, passionflower, witch hazel, ginger or Echinacea, or single active compounds thereof;
(v) α-bisabolol, apigenin, apigenin-7-glucoside, gingerol, shogaol, gingerdiol, dehydrogingerdione, paradol, natural avenanthramides, unnatural avenanthramides, preferably dihydroavenanthramide D, boswellic acid, phytosterols, glycyrrhizin, glabridin and licochalcone A, preferably in the form of pure substances;
(vi) preferably sodium lactate, urea and derivatives, glycerol, propylene glycol, 1,2-pentanediol, 1,2-hexanediol and 1,2-octanediol, collagen, elastin or hyaluronic acid, diacyladipates, petrolatum, urocanic acid, lecithin, allantoin, panthenol, phytantriol, lycopene, (pseudo)ceramides (preferably ceramide 2, hydroxypropyl bispalmitamide MEA, cetyloxypropyl glyceryl methoxypropyl myristamide, N-(1-hexadecanoyl)-4-hydroxy-L-proline (1-hexadecyl) ester, hydroxyethyl palmityloxyhydroxypropyl palmitamide), glycosphingolipids, glyceryl hydroxypropyl ester ... skin care agents, preferably skin moisture regulating or skin repairing agents, selected from the group consisting of chondroitin sulfates, cholesterol, phytosterols, chitosan, chondroitin sulfate, lanolin, lanolin esters, amino acids, vitamin E and derivatives (preferably tocopherol, tocopheryl acetate), α-hydroxy acids (preferably citric acid, lactic acid, malic acid) and derivatives thereof, mono-, di- and oligosaccharides, preferably glucose, galactose, fructose, mannose, fruit sugar and lactose, polysaccharides, such as β-glucans, in particular 1,3-1,4-β-glucan from oats, α-hydroxy-fatty acids, triterpene acids, such as betulinic acid or ursolic acid, and algae extracts or single active compounds thereof,
(vii) preferably menthone glycerol acetal, menthyl lactate, preferably 1-menthyl lactate, in particular 1-menthyl 1-lactate), menthyl ethyl oxamate, substituted menthyl-3-carboxylic acid amides (e.g. menthyl-3-carboxylic acid N-ethylamide, Nα-(L-menthanecarbonyl)glycine ethyl ester, 2-isopropyl-N-2,3-trimethylbutanamide, substituted cyclohexanecarboxylic acid amides, 3-menthoxypropane-1,2-diol, 2-hydroxyethyl menthyl carbonate, 2-hydroxypropyl menthyl carbonate, (viii) physiological cooling agents selected from the group consisting of: benzoates, N-acetylglycine menthyl esters, isopulegol, menthyl hydroxycarboxylates (e.g. menthyl 3-hydroxybutyrate), monomenthyl succinate, monomenthyl glutarate, 2-mercaptocyclodecanone, menthyl 2-pyrrolidin-5-one carboxylate, 2,3-dihydroxy-p-menthane, 3,3,5-trimethylcyclohexanone glycerol ketal, 3-menthyl 3,6-di- and trioxaalkanoates, 3-menthyl methoxyacetate and icilin, and (viii) histamine receptor antagonists, serine protease inhibitors, TRPV1 antagonists, NK1 antagonists, cannabinoid receptor agonists and TRPV3 antagonists,
is selected from the group consisting of:

According to a further aspect, the present invention also relates to the cosmetic use of a microorganism or a mixture comprising or consisting of two microorganisms for topical application to the skin, in particular to improve the appearance of the skin and/or to prevent body odor, the microorganism being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), preferably the microorganism being an attenuated or killed microorganism that has been subjected to a heat treatment and/or has an intact physical structure.

"Cosmetic use" is non-therapeutic use, i.e. use on skin whose appearance is affected, for example, by irritation, redness, dryness, flaking, rashes, signs of aging or minor acne, and which represents a cosmetic problem or a slight discomfort, but is not characterized as pathological. A further "cosmetic use" is the prevention or reduction of body odor associated with the proliferation of certain microorganisms on the skin that cause malodour but do not represent an immediate danger to the patient's health.

The above cosmetic uses are therefore preferably for reducing or preventing skin irritation, dry skin, rashes, acne, body odor and/or skin aging.

In the cosmetic use according to the invention, the microorganisms are
(a) agents that strengthen the skin barrier function, and/or (b) agents that reduce transepidermal water loss, and/or (c) agents that induce the expression of filaggrin, and/or (d) agents that increase components related to natural moisturizing factors, and/or (e) agents that provide anti-inflammatory activity, in particular agents that reduce and/or suppress inflammatory parameters, and/or (f) agents that provide anti-inflammatory activity induced by chemokines, and/or (g) agents that provide anti-inflammatory activity induced by external pathogenic toxins, such as pathogenic microorganisms, in particular Staphylococcus aureus, and/or air pollutants, and/or ultraviolet light and/or surfactants, and/or (h) agents that inhibit the growth and/or invasion and/or infection of pathogenic microorganisms, in particular Staphylococcus aureus, and/or (i) agents that induce the expression of antimicrobial peptides (AMPs), and/or (j) agents that maintain and/or establish and/or restore the health of the skin microbiome, and/or (k) agents that improve the immune response of the skin,
It is used as.

The effects described in items (a)-(k) and further described in more detail above also represent suitable combinations that address multiple problems related to cosmetics alone. For example, a reduction in the skin barrier function can lead to increased transepidermal water loss resulting in dry skin that shows signs of irritation such as redness or flaking, thus affecting the appearance of the skin. Upregulation of filaggrin and an increase in components related to natural moisturizing factors prevents or reduces such problems. Anti-inflammatory activity reduces the signs of swelling or redness caused by skin reactions to certain stimuli that affect the appearance of the skin but do not represent pathological symptoms. Inhibiting the growth of pathogenic microorganisms, for example by inducing the expression of AMPs, can eliminate mild cases of acne or prevent and reduce body odor.

In a preferred embodiment of the above cosmetic use, the microorganism is used in the form of a preparation according to any of the above embodiments.

In a further aspect, the present invention relates to a cosmetic method for improving the appearance of the skin and/or preventing body odor, in particular to reduce or prevent skin irritation, dry skin, rashes, acne, body odor and/or skin aging, comprising the steps of:
i) topically applying to the skin a microorganism or a mixture comprising or consisting of two microorganisms, wherein the microorganism is selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), or applying to the skin a preparation or a cosmetic composition or product as described above, preferably wherein the microorganism is an attenuated or killed microorganism that has been subjected to a heat treatment and/or has an intact physical structure,
The present invention also relates to a method comprising the steps of:

A "cosmetic method" is a non-therapeutic method that solves a cosmetic problem but does not treat a pathological condition, as described in more detail above in the context of cosmetic use. In the cosmetic method according to the present invention, the microorganism acts as an agent that provides the above-mentioned effects in items (a)-(k), and thus can solve multiple cosmetic problems described above in the context of cosmetic use.

Figure 1 shows induction of antimicrobial peptides by L. plantarum HEAL19 in HaCaT keratinocytes. Significantly induced genes are β-defensin 3, β-defensin 4, peptidase inhibitor 3, RNase 7, psoriasin, lactotransferrin and secretory leukocyte peptidase inhibitor. Gene expression of antimicrobial peptides in HaCaT keratinocytes was measured after 24 hours treatment with 0.05% L. plantarum HEAL19 using gene arrays. Induction was defined as an RQ-calculated value of >2. Figure 1 shows concentration-dependent induction of β-defensin 3 by L. plantarum HEAL19 in HaCaT keratinocytes. HaCaT keratinocytes were treated with various concentrations of L. plantarum HEAL19 for 24 hours, resulting in RQ-calculated values ranging from 243.6 (0.1% L. plantarum HEAL19) to 2.3 (0.0125% L. plantarum HEAL19). Figure 1 shows the differential induction of AMP coding genes after 24 hours of treatment. RNase 7, β-defensin 3 peptidase inhibitor 3, phospholipase A2, and secretory leukocyte peptidase inhibitor are induced in a concentration-dependent manner (0.1 and 0.05% were tested) by L. plantarum HEAL99. Anti-inflammatory effect of L. plantarum HEAL 19 and 99 with dexamethasone included as positive control. Confluent HaCaT keratinocytes were pretreated with test materials 0.1 mg/ml L. plantarum HEAL 99, 0.25 mg/mL L. plantarum HEAL 19 and 30 μM dexamethasone. After 48 hours of incubation, keratinocytes were stimulated with 30 ng/mL of inflammatory IL-1α for 8 hours. Subsequent interleukin 8 release from HaCaT keratinocytes due to IL-1α stimulation after pretreatment with L. plantarum HEAL 19 and HEAL 99 was measured in cell culture medium using ELISA. Comparison with the positive control dexamethasone and non-pretreated keratinocytes (IL-1α control) is shown. Figure 1 shows IL-8 release (ordinate) after treatment of an ex vivo human skin model with placebo and L. plantarum HEAL19 preparations and stimulation with diesel particulate 1650b (simulating air pollution). Figure 1 shows interleukin-8 release from HaCaT keratinocytes due to S. aureus stimulation after pretreatment with L. plantarum HEAL 19. Comparison with non-pretreated keratinocytes is shown (S. aureus control). 1 shows the growth inhibitory effect of S. aureus when treated with L. plantarum HEAL19 in a dose-dependent manner, i.e., 0.1 and/or 0.05%. Comparison with a positive growth control and a negative control (inhibition control, addition of the biocide farnesol) is shown. Figure 1 shows the production of involucrin (ordinate) in an ex vivo human skin model after treatment with various concentrations of L. plantarum HEAL19 (x-axis), as compared to a negative control containing only the vehicle (DMSO). Figure 1 shows the production of cytokeratin 14 (CK14) content (ordinate) in an ex vivo human skin model after treatment with various concentrations of L. plantarum HEAL19 (x-axis), as compared to a negative control containing only the vehicle (DMSO). Percentage of Rhodamine B detection (ordinate) is shown following in vitro treatment with placebo and L. plantarum HEAL19 preparations and challenge with diesel particulate 1650b. Figure 1 shows the length (nm) of intercellular lipid lamellae per 1000 nm2 of intercellular space measured in a 3D epidermal model system for dry skin. The lamellae were disrupted due to SDS treatment. After treatment with 0.5% L. plantarum HEAL19 in a skin care formulation, the skin repair performance was determined by transmission electron microscopy (TEM). Figure 1 shows the hyaluronic acid release (ordinate) of the HEPKp 3D skin model after treatment with L. plantarum HEAL19 and HEAL99. A comparison is shown with the negative control containing only cell culture medium (medium control), x-axis. Figure 1 shows filaggrin release (ordinate) from the HEPKp 3D skin model after treatment with L. plantarum HEAL19 and HEAL99. Comparison is shown with the negative control containing only cell culture medium (medium control), x-axis. 1 shows filaggrin content in an ex vivo human model after treatment with two different concentrations of L. plantarum HEAL19. Comparison with a negative control containing only vehicle (DMSO) is shown. Figure 1 shows the moisture content in human skin in vivo after treatment with L. plantarum HEAL19 at a concentration of 1% in a cosmetic cream, as determined by capacitance. The grey shaded bars represent the cream with L. plantarum HEAL19, the white bars represent the placebo cream, and the black bars represent no treatment. Figure 1 shows the skin barrier strength in human skin in vivo after treatment with L. plantarum HEAL19 at a concentration of 1% in a cosmetic cream, as determined by transepidermal water loss (TEML). The grey shaded bars represent the cream with L. plantarum HEAL19, the white bars represent the placebo cream, and the black bars represent no treatment. Growth curve of L. plantarum in MRS medium over 16 hours. Start - OD = 0.1; average of 3 cavities.

The present invention is further illustrated by the following examples, which should not be construed as limiting in scope.

The following experimental procedures were used in the examples:

Preparation of heat-treated Lactobacillus suspension

Growth of L. plantarum strains HEAL19 and HEAL99 can be obtained in various culture media. The carbon source can be glucose or starch. Meat extract, yeast extract, peptone or protease peptone (vegetable), etc. can be used as nitrogen source. The pH of the medium should be about 6. The culture temperature can vary but is preferably 37°C. The culture time can be about 8-24 h. Shaking or aeration shaking can be added.

A washing process can be added after the culture. It can be carried out as follows: first, the cells can be harvested by centrifugation, the supernatant can be decanted and the cell pellet can be suspended in liquid PBS, cell culture medium or purified water. The described procedure can be repeated if necessary.

A heat treatment of the Lactobacillus suspension can be carried out before or after the washing procedure. It can be achieved by ultrasound, UV irradiation or heat, but heat treatment is preferred. It can be carried out at a temperature between 60°C and 121°C. For temperatures above 100°C an autoclave may be used. The heat treatment time can range from 1 second to 120 minutes, for example 20 seconds to 120 minutes. Heat treatment between 70 and 100°C for 20 seconds to 15 minutes using an industrial pasteurizer is preferred. Optional further processing for the heat treated Lactobacillus may include freeze drying, spray drying, granulation, etc. This may serve the purpose of improving the usability and/or stability of the product itself.

The final product may be a powder, granules, suspension or solution and is intended to be applied at a concentration of ^0.001-10 % weight/volume in the cosmetic composition, which corresponds to a concentration of ^1x107-1x1011 cells/mL of the cosmetic formulation. More preferably, the final product is applied at a concentration of 0.01-5%, even more preferably 0.02-1% weight/volume, for example 0.1-1% weight/volume.

Effect of Lactobacillus strains on AMP gene expression in keratinocytes grown as monolayers

AMP induction was measured by real-time PCR analysis using TaqMan® Array Rapid 96-well plates.

HaCaT keratinocytes were cultured in EpiLife medium (cascade Biologics, Gibco) with HKGS (Gibco Technologies). Test substances were diluted in an equal volume of medium and added to 6-well plates at a volume of 2 mL per well. All samples were tested on 100% confluent cells incubated at 37°C and 5% CO2. After 24 h incubation time, all samples were removed and cells were washed twice with Dulbecco's phosphate buffer with Ca and Mg (Capricorn). RNA isolation was performed using RNeasy® Mini Kit (Qiagen). This procedure was carried out as described in the RN Easy Mini Handbook.

Cells were lysed and precipitated with ethanol, the homogenate was washed three times using spin columns, and the mRNA was eluted with purified water. This procedure was slightly adjusted depending on the sample.

RNA concentration was measured by measuring the absorbance at 260 nm using a μCuvette G 1.0 and a BioPhotometer (Eppendorf). Control values such as E260/280 or E260/230 were calculated simultaneously. Transcription was performed with a minimum amount of 0.5-1 μg RNA per sample.

Reverse transcription was performed using the RNA to cDNA High Capacity Kit from Applied Biosystems. The procedure was based on the provided protocol. All samples were incubated in a PCR Thermocycler (Biometra) running 37°C for 60 min for reverse transcription followed by 95°C for 5 min (enzyme inactivation) and maintaining the temperature in the reaction chamber at 4°C.

Quantitative real-time PCR was performed using a StepOne Plus Fast Real Time PCR machine (Applied Biosystems). The initial steps of RT-PCR included a first heating step at 95°C for 20 s, followed by 40 cycles of cDNA denaturation at 95°C for 3 s and annealing/extension at 60°C for 30 s.

The analysis was carried out by the 2-ΔCT method. The details are as follows.
1. Normalization of Ct values using the reference-gen (HPRT) ΔC _T value _gene = C _T value _gene - C _T value _{reference gene (HPRT)}
2. Subtraction of the ΔC _T -value of the control sample (untreated) from the ΔC _T -value (treated) ΔΔC _T value _gene = ΔC _T value _treated - ΔC _T value _control
3. Calculation of RQ-ratio (relative quantitative value) RQ-value = 2 ^-ΔΔCT

An RQ-value of >2 was defined as valid gene induction.

Anti-inflammatory effects of Lactobacillus plantarum HEAL19 and HEAL99

HaCaT keratinocytes were cultured in 96-well plates with RPMI-A medium, filter sterilized, supplemented with L-glutamine (Capricorn) and 10% fetal calf serum (FCS) (Capricorn). Plates were incubated at 37°C and 5% CO2. After 24 hours of starvation with cell culture medium (RPMI, 1% FCS), cells were washed once. Anti-inflammatory substances/inactivated bacteria suspensions were suspended in starvation culture medium and applied and incubated for 24/48 hours with the above parameters. Dexamethasone at a concentration of 10 μM was used as a positive control. After pre-incubation all wells were washed 3 times with cell culture medium to remove bacterial cells. IL-1α (Gibco Life Technologies) was dissolved in distilled _H2O , diluted in starvation culture medium and pipetted into the wells at a concentration of preferably 30 ng/mL. Alternatively, heat-inactivated Staphylococcus aureus (DSM 799) suspension was used as an inducing agent at concentrations between 0.1 mg/mL and 1 mg/mL dry weight. After 8 hours of incubation, the supernatant was separated. All samples were stored at -80°C until further analysis.

IL-8 concentrations were measured with a human CXCL8/IL-8, DuoSet ELISA (Development Systems, R&D Systems). Experimental procedures were performed as described in the provided protocol.

After calculating the mean of duplicate/triplicate values and subtraction of the mean zero standard optical density, a four-parameter logistic curve fit was used to generate the standard curve.

Hyaluronic acid induction

Human epidermal keratinocyte precursor (HEPKp)-generated 3d human skin models were cultured and treated systemically with test substances. Briefly, the test substances at defined concentrations were added to the cell culture medium, and each skin model was treated in cell culture dishes at 37°C and 5% ^CO2 for up to 9 days. After treatment, the collected supernatants were stored at -80°C until further analysis. Cell viability was controlled by the MTT standard method.

Hyaluronic acid concentrations were measured by TECOmedical Hyaluronic Acid Plus ELISA. Experimental procedures were performed as described in the provided protocol.

Antibacterial effects of Lactobacillus strains

Growth curves of various bacteria were measured photometrically at 620 nm.

Inhibition of Staphylococcus aureus:
96-well MTPs were prepared according to DIN 58940-8 with autoclaved CASO medium (MERCK 1.05459) and test substances dissolved in DMSO or distilled water. Relevant wells were inoculated with S. aureus (DSM 799/ATCC 6538) using stock cultures maintained at -20°C in 10% glycerol. An inoculum of 1-6x106 ^cfu was added per well. Test substances were analysed in triplicate or quadruplicate wells upon 16h incubation at 37°C. Growth curves for each well were determined by absorbance at 620 nm using a Sunrise Photometer (Tecan, Austria) and Magellan software. Concentrations of 125 ppm farnesol and 4 ppm triclosan, both pre-dissolved in DMSO (Merck 802912), were used as positive controls for growth inhibition.

For calculations, the average absorbance detected was calculated and normalized using the blank value. For graphical representation, these OD values were plotted as a function of time.

Induction of moisturizing factor filaggrin in a 3D skin model

Three-dimensional skin models derived from human epidermal keratinocyte precursors (HEPKp) were treated systemically with test substances in CnT-PR-3D medium for 9 days. Filaggrin release in the medium was detected by ELISA (Enzyme-linked Immunosorbent Assay) kit for filaggrin (Cloud-Clone Corp.).

Barrier improvement efficacy on SDS-damaged 3D skin model for dry skin

For this purpose, in vitro studies were performed on a 3D epidermal model system for dry skin. The model system consisted of 14-day-old mature epidermal cell cultures with a perturbed epidermal skin barrier due to SDS treatment. Perturbation of the epidermal barrier was measured as a reduction in intercellular lipid lamellae. Skin repair performance was determined by transmission electron microscopy (TEM) after treatment with L. plantarum HEAL19 formulated in a cosmetic emulsion or a placebo emulsion and then further cultured to observe the reformation of the intercellular lipid lamellae.

In vitro measurements

In vitro trials were performed by Cutech Srl (Italy). The effect of the described Lactobacillus strains on various parameters was analyzed in an in vitro skin model. Human skin from abdominal plastic surgery was used. Tape stripping followed by systemic or topical application of heat-inactivated bacterial suspensions in formulations or dissolved in cell culture medium may be used. Concentrations and incubation times may be varied.

All data were processed for mean, standard deviation and standard error (SEM) for each treatment.

Epidermal filaggrin, involucrin and cytokeratin 14
For analysis, models were immunostained with selected antibodies filaggrin rabbit polyclonal [H-300], involucrin mouse monoclonal (SY5) and/or cytokeratin 14 (CK14) rabbit monoclonal (EPR17350). The amount of antigen present in each slide was assessed by evaluating the intensity and distribution of red pigment within the epidermis. The data obtained were normalized for the length of the basal layer.

Skin Barrier Integrity (Rhodamine B)
Ex vivo skin models were topically treated with L. plantarum strains prior to application of diesel particulates (i.e. 1650b). All skin samples were incubated at 35°C, 5% CO2 and ambient humidity. At the end of this experimental phase, skin samples were collected, stained with Rhodamine B, cryofixed and cut in a cryostat for image acquisition and analysis of the results. Analysis of Rhodamine B fluorescence was performed in two sections of the epidermal area for each skin model. Images were analyzed by evaluating fluorescence with Image-J application (NIH, USA).

Anti-inflammatory Effects The in vitro human skin model described above was used to confirm the anti-inflammatory potential of L. plantarum strains. The model was treated with L. plantarum HEAL19 cosmetic emulsion followed by application of pollutant 1650b, which simulates external toxic agents and air pollution. At selected end points, organotypic cell culture medium was aspirated from the wells and assayed for IL-8 using Biolegend® Inc. Deluxe set human IL-8.

In vivo measurements

In vivo trials were conducted at Kosmoscience Ciencia & Technologia Cosmetica Ltda, Brazil. They evaluated relevant skin parameters after application of a cosmetic oil/water formulation containing L. plantarum HEAL19 lyophilisate.

The oil/water emulsion had the following composition:

Twenty-two subjects with severe dry skin participated in this study. Topical application of the emulsion was performed twice daily on the inner forearm. Measurements were taken on days 0, 7, 14, and 21.

Skin Hydration by Corneometry Capacitance measurements were performed using a Corneometer® 825 probe coupled with a Multi Probe Adapter MPA5 (CKelectronis, Germany). The variation of capacitance and skin hydration provided by the L. plantarum HEAL19 formulations versus placebo was calculated.

Skin barrier efficacy by evaporometry Transepidermal water loss (TEWL) was measured using a Tewameter® 300 probe coupled with a multi-probe adapter MPA5 (CK Electronics, Germany). The variation of TEWL values and transdermal barrier enhancement provided by the L. plantarum HEAL19 formulations relative to placebo was calculated. Enhancement of the skin barrier can be observed as a decrease in TEWL values.

The following results demonstrate the effectiveness of the present invention. Each individual experiment should illustrate but not limit the effectiveness of the present invention.

Example 1: AMP gene expression in HaCa keratinocytes L. plantarum HEAL19 induces the release of a wide range of antimicrobial peptides in human keratinocytes. All these peptides together represent the complex defense system of human skin. For example, defensins that can kill gram-positive bacteria were upregulated. In summary, the upregulated peptides are effective against a wide range of potentially pathogenic gram-positive and gram-negative bacteria, fungi, eukaryotic parasites and/or viruses.

Figure 1 shows gene expression of antimicrobial peptides in HaCaT keratinocytes after 24 h treatment with 0.05% L. plantarum HEAL19. Gene expression was measured using gene arrays. Induction was defined as a calculated RQ-value >2.

The results show that treatment with L. plantarum HEAL19 induces important AMPs such as β-defensins 1, 3 or 4 and/or psoriasin and/or calprotectin. This treatment results in a higher expression of AMPs in human skin that are associated with a stronger first line of defense in human skin, thereby supporting the skin barrier. Furthermore, induced expression of β-defensins or cathelicidins can balance the AMP fingerprint in diseases such as in atopic dermatitis. Thus, treatment with L. plantarum HEAL19 can restore, maintain and/or improve the skin microbiome.

The induction of AMP by L. plantarum HEAL19 is concentration-dependent. Figure 2 shows the concentration-dependent induction of β-defensin 3. HaCaT keratinocytes were treated with various concentrations of L. plantarum HEAL19 for 24 hours. This results in calculated RQ values ranging from 243.6 (with 0.1% L. plantarum HEAL19) to 2.3 (with 0.0125% L. plantarum HEAL19).

Similar effects can be observed with L. plantarum HEAL99 when treating HaCaT keratinocytes. Figure 3 shows an exemplary differential induction of AMP-encoding genes after 24 hours of treatment. RNase 7, β-defensin 3 peptidase inhibitor 3, phospholipase A2, and secretory leukocyte peptidase inhibitor are induced in a concentration-dependent manner by L. plantarum HEAL99 (0.1 and 0.05% were tested). Similar results were found with L. plantarum Heal19.

In an epidermal 3D human skin model, various AMP-encoding genes were shown to be similarly induced (data not shown).

Example 2: Anti-inflammatory effects of Lactobacillus strains (in vitro/ex vivo)
Skin disease, injury or irritation is mostly correlated with inflammation of the affected skin.In addition to the described beneficial effects of Lactobacillus strains, their topical application has been shown to result in anti-inflammatory effects.In this regard, this Lactobacillus strain supports skin health through another mechanism.

Figure 4 shows the anti-inflammatory effect of L. plantarum HEAL 19 and 99 with dexamethasone included as a positive control. Confluent HaCaT keratinocytes were pretreated with the test materials 0.1 mg/mL L. plantarum HEAL 99, 0.25 mg/mL L. plantarum HEAL 19 and 30 μM dexamethasone. After 48 hours of incubation, keratinocytes were stimulated with 30 ng/mL of the proinflammatory IL-1α for 8 hours. The subsequent release of IL-8 in the cell culture medium was measured using ELISA. Application of Lactobacillus strains allowed a reduction of HaCaT IL-8 release to 12.6% and 5.1% (Heal 99 and Heal 19), respectively, compared to the non-pretreated IL-1α control. These anti-inflammatory effects were similar to those of dexamethasone (reduction to 20.4%), demonstrating the anti-inflammatory potential of these two strains and highlighting their potential for use in skin palliatives.

It has been shown that a similar anti-inflammatory effect of L. plantarum HEAL19 can be seen in an in vitro human skin model. As shown in Figure 5, after stimulation with diesel particulates (1650b) simulating air pollution, IL-8 detection in the cell culture medium was reduced by 75% in the L. plantarum HEAL19-treated model compared to the placebo control. Topical treatment with a cosmetic formulation containing L. plantarum HEAL19 causes an improvement in the barrier integrity of the skin, and the skin is soothed and relieved.

Inflammation in the skin can be induced not only by proinflammatory cytokines but also by bacteria, specifically bacteria pathogenic to the skin, by cell-to-cell contact. Figure 6 shows that HaCaT keratinocytes pretreated for 48 hours with 0.25 mg/mL L. plantarum HEAL19 rapidly reduces S. aureus-induced inflammation. IL-8 concentration in the medium can be reduced to 31% compared to the control stimulated with S. aureus alone.

S. aureus is frequently found in the eczematous skin lesions of AD patients and is often responsible for exacerbating the disease condition and/or promoting inflammation. Treatment of the skin with L. plantarum strains results in a reduction in inflammation, which in turn reduces redness and leaves the skin feeling soothed and calmed.

Example 3: Antibacterial effect of Lactobacillus plantarum HEAL19 and HEAL99 For example, S. aureus is consistently found in eczematous skin lesions of AD patients and is known to be important and causative for the progression of this disease state. Application of L. plantarum strains to the skin can prevent potential infection of the skin by inhibiting the growth of pathogens such as S. aureus.

A growth curve of S. aureus was generated by measuring the optical density at 16 hours of incubation. Figure 7 shows the growth inhibitory effect of S. aureus when treated with 0.1 and/or 0.05% L. plantarum HEAL19. In conclusion, topical treatment of the skin with the described invention can contribute to reducing and/or preventing inflammation and infection by inhibiting the growth of S. aureus.

Example 4: Barrier reinforcement
A: Induction of involucrin and cytokeratin 14 (CDK14) in vitro assays . Involucrin is a protein that provides structural support to skin cells, allowing them to resist invasion by microorganisms. Cytokeratin 14 is usually found as a heterodimer and forms the cytoskeleton of epidermal cells. Both marker proteins were increased by topical treatment with various concentrations of L. plantarum HEAL19 in vitro regenerated human skin models as shown in Figures 8 and 9.

This demonstrates that topical treatment of the skin with the described invention directly leads to enhanced skin barrier function.

B: Skin barrier health The present invention shows protective activity on skin barrier by reducing rhodamine B penetration in an in vitro model after treatment with L. plantarum HEAL19 and stimulation with diesel particulates (1650b). Evaluation of skin morphology allows the determination of whether a compound affects the structure of treated skin samples. To carry out this evaluation, skin sections were stained with rhodamine B stain. After staining each skin sample, epidermal health was evaluated. Figure 10 shows that treatment with L. plantarum HEAL19 improves skin health and has protective activity on skin barrier function.

C: Barrier improvement efficacy The intercellular lipid lamellae in the intercellular space of the stratum corneum were analyzed and quantitatively evaluated as an index of the quality of the epidermal barrier. Topical treatment with 0.5% L. plantarum HEAL19 in skin care formulations results in an increase in the repair performance of a 3D epidermal model system of dry skin (Figure 11). Topical application of skin care formulations containing the present invention enhances epidermal barrier function and supports the repair performance of the skin.

Example 5: Hyaluronic Acid Derivation in a 3D Skin Model Hyaluronic acid (HYA) plays a series of important roles in the skin, where it is necessary to maintain the epidermal barrier function and the structure of the stratum corneum. Furthermore, it plays an important role in fixing water in tissues, tissue repair or influencing cell differentiation and proliferation (Weindl et al., 2004). All this can be achieved by treatment with the described L. plantarum strain.

Figure 12 shows increased release of hyaluronic acid in the HEPKp 3D skin model after 8 days of treatment with 0.005% L. plantarum HEAL19 or HEAL99 in cell culture medium. Compared to the medium control, hyaluronic acid release increased to 145 and 115%.

Example 6: Skin Hydration
A: Filaggrin release from 3D skin models. Increased release of the protein filaggrin into the medium could be detected after 3-9 days of incubation. Figure 13 shows increased release of filaggrin (FLG) in the medium of a Lactobacillus-treated HEPKp 3D skin model after 8 days. At the concentration used of 0.05% L. plantarum HEAL19 and HEAL99, FLG increased to 215 and 150% in the medium, respectively.

Topical application of the described invention results in filaggrin upregulation in human skin, with subsequent positive effects on strengthening the skin barrier and skin moisturization.

B: In vitro filaggrin induction In a study using an in vitro human skin model, the induction of filaggrin due to topical treatment according to the present invention was confirmed. Figure 14 shows the concentration-dependent induction of filaggrin release after treatment with various concentrations of L. plantarum HEAL19. The higher the applied concentration, the higher the filaggrin release in the in vitro human skin model.

A similar concentration-dependent effect was observed in in vitro experiments examining the HEPKp3D human skin model (data not shown).

Example 7: In vivo data To confirm the effects observed in the model system, an in vivo study was conducted with a panel of subjects with severe dry skin symptoms. Heat-treated L. plantarum HEAL19 was incorporated into a cosmetic cream and applied to the inner forearm of the panelists. The effects on skin moisturizing capacity (capacity) and transepidermal water loss (TEWL) were observed after 0, 7, 14 and 21 days compared to untreated and placebo-treated areas.

This in vivo study confirmed the effects suggested by the molecular biology studies. Capacitance steadily increased with treatment time, and the effect was significantly stronger than that of placebo treatment at all time points. TEWL was significantly reduced in the treated Lactobacillus-containing cream, whereas the placebo had no effect.

These results demonstrate the ability of the heat-treated Lactobacillus strains of the present invention to increase skin hydration and barrier strength, resulting in significant improvements in skin health.

Figures 15 and 16 show moisture content and skin barrier strength over time as determined by capacitance and TEWL.

Example 8: Growth curves of L. plantarum 96-well MTPs were prepared using autoclaved MRS medium (Oxoid CM0359). Each L. plantarum strain was inoculated into triplicate wells using stock cultures maintained at -20°C in 10% glycerol. An inoculum of OD = 0.1 was added per well and the 96-well MTPs were incubated at 37°C for 16 hours. Growth curves for each well were determined by absorbance at 620 nm using a Sunrise photometer (Tecan, Austria) and Magellan software. Saline (0.9% sodium chloride solution) was used as a negative growth control.

For calculation, the average absorbance values detected were calculated and plotted as a function of time for graphical representation. The strains tested as well as the growth curves obtained can be seen in FIG.

Formulation Examples 1-15
Formulations (compositions) comprising Lactobacillus according to the present invention having skin soothing and barrier strengthening effects:
1. Skin Lightening Day Cream Oil/Water 2. Skin Soothing Lotion 3. After Sun Balm, Reduces Itching 4. Calming Body Spray 5. Sunscreen Lotion (Oil/Water, Broad Spectrum Protection)
6: Oil/water night cream 7: Scalp soothing anti-dandruff shampoo 8: Self-tanning cream 9: Anti-itch barrier repair cream 10: Antiperspirant/deodorant roll-on 11: Emulsion with UVA/B broadband protection 12: Sun spray with UVA/B broadband protection with low oil content 13: Skin lightening balm with UVA/UVB 14: Scalp soothing hair conditioner with UVB/UVA protection, rinse off 15: Anti-itch hair conditioner, leave-on

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Yet another aspect of the present invention may be as follows.
[1] A microorganism or a mixture containing or consisting of two types of microorganisms for use in the treatment and/or prevention of skin conditions by topical application, wherein the microorganism is selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), or a mixture containing or consisting of two types of microorganisms.
[2] The microorganism or mixture for use according to [1] above, wherein the microorganism has been subjected to heat treatment and is preferably an attenuated or killed microorganism with an intact physical structure.
[3] The microorganism or mixture for use according to [1] or [2] above, wherein the skin symptom is a decrease in skin barrier function, an inflammatory skin symptom and/or the proliferation of pathogenic microorganisms, and preferably the skin symptom is selected from the group consisting of atopic dermatitis, microbial infection, dry skin, skin itching, sensitive skin, atopic skin, skin inflammation, microbial dysbiosis, rosacea, psoriasis, rash and acne.
[4] A preparation comprising or consisting of one or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316) and a carrier, said preparation being a particulate preparation, in particular a granule or powder.
[5] The preparation according to [4] above, wherein the microorganism has been subjected to heat treatment and is preferably an attenuated or killed microorganism with an intact physical structure.
[6] The preparation according to [4] or [5], wherein the carrier is selected from the group consisting of inulin, starch, gum arabic, whey protein, skim milk powder and maltodextrin, and combinations thereof, preferably maltodextrin.
[7] The preparation according to any one of [4] to [6], wherein the ratio of microorganism to carrier is in the range of 1:9 to 3:7, preferably 1.5:8.5 to 2.5:7.5, and/or the preparation comprises 10 to 30% by weight of microorganism and 70 to 90% by weight of the carrier, preferably 15 to 25% by weight of microorganism and 75 to 85% by weight of the carrier, in each case relative to the total weight of the preparation.
[8] A method for producing a preparation comprising or consisting of one kind of microorganism or a mixture of two kinds of microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM15313) and Lactobacillus plantarum HEAL99 (DSM15316) and a carrier, preferably the preparation according to any one of [4] to [7] above, comprising:
(i) providing a microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316);
(ii) optionally subjecting the microorganism of step (i) to a heat treatment, preferably at a temperature in the range of 60 to 121° C. for a period of 1 second to 120 minutes;
(iii) combining the microorganism from step (i) or (ii) with a carrier and further processing the combination, preferably by freeze-drying, spray-drying or granulation, to obtain a granule or powder;
The method includes:
[9] A pharmaceutical or cosmetic composition or pharmaceutical or cosmetic product for topical application to the skin, comprising one or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM15313) and Lactobacillus plantarum HEAL99 (DSM15316) or comprising the preparation according to any one of [4] to [7] above, wherein the total amount of the microorganisms is sufficient to treat and/or prevent skin conditions, in particular inflammatory skin conditions, impaired skin barrier function, and/or the proliferation of pathogenic microorganisms, preferably the total amount of the microorganisms is in the range of 0.01 to 5% by dry weight, preferably 0.02 to 1% by dry weight, in each case relative to the total weight of the composition, and/or the total amount of the microorganisms is at least 10 ⁸ , preferably 5 x 10 ⁸ cells or at least 10 ¹¹ cells, for example in the range of 10 ⁸ to 5×10 ¹⁰ , preferably 10 ⁹ to 10 ¹⁰ cells per gram of said total composition.
[10] The pharmaceutical or cosmetic composition or product according to [9], wherein the composition or product is selected from the group consisting of oil-in-water or water-in-oil emulsions, ointments, cremes, lotions and gels, and preferably the composition or product further comprises one or more components selected from the group consisting of carriers, excipients and further active ingredients.
[11] A method for producing a pharmaceutical or cosmetic composition or product, preferably the pharmaceutical or cosmetic composition or product described in [9] or [10], comprising the steps of:
(i) preparing a mixture of one or two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM15313) and Lactobacillus plantarum HEAL99 (DSM15316), preferably the microorganism is subjected to heat treatment, preferably an attenuated or killed microorganism with an intact physical structure, or preparing a preparation according to any one of [4] to [7] above or a preparation produced by the method according to [8] above;
(ii) combining said microorganism or said preparation of step (i) with one or more substances selected from carriers, excipients and further active ingredients to obtain a pharmaceutical or cosmetic composition or product;
The method includes:
[12] Cosmetic use of a microorganism or a mixture containing or consisting of two microorganisms for topical application to the skin, in particular to improve the appearance of the skin, said microorganism being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), preferably said microorganism being an attenuated or killed microorganism that has been subjected to a heat treatment and/or has an intact physical structure.
[13] The cosmetic use according to [12] above for reducing or preventing skin irritation, dry skin, rashes, acne, and/or skin aging.
[14] The cosmetic use according to [12] or [13], wherein the microorganism is used in the form of a preparation according to any one of [4] to [7] or a preparation produced by the method according to [8].
[15] A cosmetic method for improving the appearance of the skin, in particular reducing or preventing skin irritation, dry skin, rashes, acne and/or skin aging, comprising:
i) topically applying to the skin a microorganism or a mixture containing or consisting of two microorganisms, said microorganism being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316); or applying to the skin a preparation according to any one of [4] to [7], or a preparation produced by the method according to [8], or a cosmetic composition or product according to [9] or [10], preferably, said microorganism is an attenuated or killed microorganism that has been subjected to a heat treatment and/or has an intact physical structure;
The method includes:

Claims (12)

1. A composition for use in the treatment and/or prevention of skin conditions by topical application to non-mucosal skin, comprising or consisting of a microorganism or a mixture comprising or consisting of two microorganisms, said microorganism being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316) , said microorganism or said mixture comprising or consisting of two microorganisms comprising at least Lactobacillus plantarum HEAL19 (DSM 15313), and said skin conditions being selected from the group consisting of dry skin, itchy skin and sensitive skin. The composition of claim 1, wherein the microorganism is a attenuated or killed microorganism that has been subjected to heat treatment and preferably has an intact physical structure. A preparation for treating and/or preventing a decrease in skin barrier function and/or the growth of pathogenic microorganisms by topical application to non-mucosal skin, comprising or consisting of one or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316) and a carrier, wherein the one or the mixture of the two microorganisms is at least Lactobacillus plantarum HEAL19 (DSM 15313) and/or Lactobacillus plantarum HEAL99 (DSM 15316). plantarum HEAL19 (DSM 15313), said preparation being a particulate preparation, in particular a granule or powder, the ratio of microorganism to carrier being in the range of 1:9 to 3:7, preferably 1.5:8.5 to 2.5:7.5, and/or said preparation comprising 10 to 30% by weight of microorganism and 70 to 90% by weight of said carrier, preferably 15 to 25% by weight of microorganism and 75 to 85% by weight of said carrier, in each case relative to the total weight of the preparation. The preparation of claim 3, wherein the microorganism is a attenuated or killed microorganism that has been subjected to heat treatment and preferably has an intact physical structure. The preparation according to claim 3 or 4, wherein the carrier is selected from the group consisting of inulin, starch, gum arabic, whey protein, skimmed milk powder and maltodextrin and combinations thereof, preferably maltodextrin. A method for producing the preparation according to any one of claims 3 to 5 , comprising the steps of:
(i) preparing a microorganism or a mixture of two microorganisms selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM15313) and Lactobacillus plantarum HEAL99 (DSM15316); wherein the microorganism or the mixture of the two microorganisms includes at least Lactobacillus plantarum HEAL19 (DSM15313);
(ii) optionally subjecting the microorganism of step (i) to a heat treatment, preferably at a temperature in the range of 60 to 121° C. for a period of 1 second to 120 minutes;
(iii) combining the microorganism from step (i) or (ii) with a carrier and further processing the combination, preferably by freeze-drying, spray-drying or granulation, to obtain a granule or powder;
The method includes: A pharmaceutical or cosmetic composition or a pharmaceutical or cosmetic product for topical application to non-mucosal skin for treating and/or preventing a compromised skin barrier function and/or the growth of pathogenic microorganisms, comprising a preparation according to any one of claims 3 to 5, wherein the total amount of said microorganisms is sufficient to treat and/or prevent a compromised skin barrier function and/or the growth of pathogenic microorganisms, preferably the total amount of said microorganisms is in the range of 0.01 to 5% by dry weight, preferably 0.02 to 1% by dry weight, in each case relative to the total weight of the composition or product, and/or the total amount of said microorganisms is at least ¹⁰⁸ , preferably ^5x108 cells or at least ¹⁰¹¹ cells per gram of the total composition or product , for example in the range of ¹⁰⁸ to ^5x1010 , preferably ¹⁰⁹ to ¹⁰¹⁰ cells per gram of the total composition or product. 8. The pharmaceutical or cosmetic composition or pharmaceutical or cosmetic product of claim 7, wherein the composition or product is selected from the group consisting of oil-in-water or water-in-oil emulsions, ointments, creams, lotions and gels , preferably wherein the composition or product further comprises one or more ingredients selected from the group consisting of carriers , excipients and further active ingredients. A method for producing a pharmaceutical or cosmetic composition or a pharmaceutical or cosmetic product according to claim 7 or 8 , comprising the steps of:
(i) providing a preparation according to any one of claims 3 to 5 or produced by the method according to claim 6 ;
(ii) combining said microorganism or said preparation of step (i) with one or more substances selected from carriers, excipients and further active ingredients to obtain a pharmaceutical or cosmetic composition or pharmaceutical or cosmetic product;
The method includes: 1. A cosmetic composition or product for topical application to non-mucosal skin to improve the appearance of skin having a skin condition selected from the group consisting of dry skin and sensitive skin , comprising or consisting of one microorganism or a mixture comprising or consisting of two microorganisms, said microorganism being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), with the proviso that said microorganism or said mixture comprising or consisting of two microorganisms comprises at least Lactobacillus plantarum HEAL19 (DSM 15313), preferably said microorganism being an attenuated or killed microorganism that has been subjected to a heat treatment and/or has an intact physical structure. The cosmetic composition or cosmetic product according to claim 10 , wherein the microorganism is used in the form of a preparation according to any one of claims 3 to 5 or a preparation produced by the method according to claim 6 . A cosmetic composition or cosmetic product comprising a preparation according to any one of claims 3 to 5 or a preparation produced by the method according to claim 6 , or a cosmetic composition or cosmetic product according to claims 7 or 8 , for use in a method for reducing or preventing skin irritation and/or dry skin, said method comprising:
i) topically applying to non-mucosal skin a microorganism or a mixture of two microorganisms, said microorganism being selected from the group consisting of Lactobacillus plantarum HEAL19 (DSM 15313) and Lactobacillus plantarum HEAL99 (DSM 15316), with the proviso that said microorganism or said mixture of two microorganisms at least comprises Lactobacillus plantarum HEAL19 (DSM 15313); or 5 or a preparation produced by the method of claim 6 or a cosmetic composition or product according to claim 7 or 8 to non-mucosal skin, preferably the microorganism is an attenuated or killed microorganism that has been subjected to a heat treatment and/or has an intact physical structure.
A cosmetic composition or product.